Growth factors play important roles in the control of cell growth, differentiation, metabolism and oncogenesis. The signals generated by a growth factor are transduced across the cellular membrane by transmembrane receptors specific for the growth factor. The diverse biological effects of growth factors are mediated by a large family of cell surface transmembrane receptors with intrinsic protein tyrosine kinase (PTK) activity. The extracellular portion of receptor PTKs contain the binding site for its particular growth factor/ligand, whereas the tyrosine kinase activity resides in the cytoplasmic portion. Binding of a growth factor to the extracellular domain of this receptor results in autophosphorylation of specific tyrosine residues in the cytoplasmic domain. These phosphotyrosines either stimulate PTK activity or serve as binding sites for downstream signalling proteins containing Src-homology 2 (SH2) or phosphotyrosine binding (PTB) domains.
Eighteen classes or subfamilies of human receptor PTKs have been identified to date, including the insulin receptor (IR), EGF-receptor, PDGF receptor and FGF-receptor. Ligand-induced dimerization of receptors such as the EGF, PDGF and FGF receptors is thought to be essential for activation. Growth factors, such as PDGF are dimeric molecules which, by themselves, are able to induce PDGF-receptor dimerization. However, FGFs are monomeric and are unable, by themselves, to induce receptor dimerization. Dimerization of FGF receptors is thought to be mediated by FGF in concert with heparin sulfate proteoglycans (soluble or cell surface bound).
In contrast to the EGF, PDGF and FGF receptors, which are monomeric and dimerize upon ligand binding, the insulin receptor exists as a "dimer." In fact, the insulin receptor is a disulphide-linked .alpha..sub.2 .beta..sub.2 heterotetramer. Binding of insulin to the extracellular .alpha.-chains is thought to cause a change within the quaternary structure of the receptor that results in autophosphorylation of specific tyrosines in the cytoplasmic portion of the .beta. chains.
In an effort to elucidate the mechanisms underlying kinase activation, the crystal structure of such proteins is often sought to be determined. The crystal structures of several protein serine/threonine kinases have been reported: cyclic-AMP-dependent protein kinase (CAPK; Knighton et al., 1994); cyclin-dependent kinase 2 (CDK2; DeBondt et al., 1993); mitogen-activated protein kinase (MAPK; Zhang et al., 1994); and twitchin kinase (Hu et al., 1994). However, the crystalline structure of only one receptor tyrosine kinase has been determined--the unphosphorylated apo form of the tyrosine kinase domain of the insulin receptor (Hubbard et al., 1994).
Despite these reports, the ability to obtain crystalline forms of the tyrosine kinase domains of non-insulin receptor tyrosine kinases; i.e., cytoplasmic tyrosine kinases and/or receptor tyrosine kinases that undergo ligand-mediated dimerization, has not been realized. A particularly illuminating example is the EGF receptor; to the Applicant's knowledge, researchers armed with the knowledge of how to obtain crystals of the tyrosine kinase domains of both the insulin receptor and serine/threonine kinases have attempted to obtain crystals of the tyrosine kinase domain of EGF receptor without success.